(a) Field of the Invention
The present invention relates to a process for separating the protein of interest from fusion protein which comprises a protein of interest and a fusion partner, and a fusion protein useful for the process.
(b) Description of the Related Art
As the genetic recombinant technology develops, the proteins which are difficult to obtain from nature can be produced in a large quantity by using fermentation of genetically engineered organism, thereby contributing to the human welfare. Hosts used for the recombinant technologies include E. coli, yeast, animal cell, etc. Especially, E. coli has the advantages for protein production in that the gene can be easily manipulated, gives high yield production, and allows use of inexpensive medium.
When a protein of interest is produced by using the recombinant E. coli, the protein can be expressed in soluble form or insoluble inclusion body, depending on their properties. The protein expressed in soluble form has its natural property due to accurate protein folding. The proteins that can coagulate together to be insoluble before forming tertiary structure do not show their natural properties. Thus, the inclusion body of insoluble protein must be solubilized according to various methods and then be refolded. In latter case, the yield and production rate of refolding process are low. Furthermore, many disulfide bonds in the insoluble protein make accurate refolding difficult. Therefore, it is advantageous to produce the protein of interest in soluble form.
When the protein of interest is expressed in soluble form, it is possible to largely reduce the number of early separation steps such as centrifugation and filtration by using expanded bed adsorption (EBA) to substantially increase the yield and productivity of the protein. To express the protein of interest in soluble form, a fusion partner which is capable to be well expressed in soluble form can be fused together with the protein of interest. The examples of fusion partner include GST, maltose binding protein, thioredoxin, ubiquitin, etc. The ubiquitin is a small peptide consisting of 76 amino acids. When the protein of interest is expressed in a fusion peptide with ubiquitin, the ubiquitin cause the protein to be well expressed in soluble form as well as to increase the expression rate, thereby making the protein in active form.
On the other hand, it is difficult to recover pure protein when the protein of interest is expressed in soluble form. When the protein of interest is expressed in insoluble inclusion body, it is easy to separate the inclusion body from soluble materials derived from E. coli such as the proteins of host cell, DNA, polysaccharides in the early stage of purification. However, it is very difficult to separate soluble protein of interest because it is mixed with the soluble contaminants such as the proteins of host cell, DNA, polysaccharides. The inclusion body can be solubilized by adding detergents such as urea before disrupting cells in order to purify the inclusion body with EBA, which loses advantage of separating insoluble inclusion body from soluble materials derived from E. coli as described above. In conclusion, when the protein of interest is expressed in a soluble form, or to be applied by EBA process, the protein of interest mixed with DNA, and polysaccharides enters subsequent purification steps. Thus, the efficient purification process is still required.
In typical purification processes, the protein of interest can be recovered from intracellular proteins by using the difference in charge, solubility, size, hydrophobicity, etc. These purification processes employ inexpensive materials, but have a low selectivity. Thus, to obtain the desired purity, many purification steps comprising various methods are required, and the purification yield decreases largely through the purification steps. In addition, these methods have a problem wherein the purification steps must be optimized to satisfy the property of the each individual protein of interest. To increase the selectivity of the protein of interest, the affinity chromatography can be commonly used where an antibody recognizing the unique structure of the protein is immobilized on resin, or a tag with affinity to specific support can be used as a fusion partner. The examples used for increasing selectivity include GST, polyhistidine, etc. (U.S. Pat. No. 5,108,919, and Korean Patent No. 177304). In case of affinity chromatography, expensive resin having affinity to the fusion partner or protein of interest restricts industrial application.
In addition, when the protein of interest is expressed in fusion protein, the fusion partner must be removed by cleavage in the subsequent step. In particular, when the protein of interest is intended for medical use, the fusion peptide must be designed to have the suitable cleavage site between the protein of interest and a fusion partner to produce an accurate N-terminus or C-terminus after cleavage, and not to have the cleavage site inside of the protein of interest. The cleavage reaction can be performed with chemical reagents such as acid and CNBr, and proteases such as Factor Xa, and enterokinase. Even though the enzymes have a relatively high selectivity, it is still possible to cleave the other site than intended cleavage site; furthermore, it has a poor efficiency and high cost.
Prior to the present invention, it was developed that a fusion peptide contained a fusion partner which was different in isoelectric point from the protein of interest, and was prepared and separated with ion exchange chromatography. U.S. Pat. No. 4,532,207 disclosed that the Arginine tag was fused with anionic EGF, and then purified through cation exchange chromatography to produce EGF. However, the method has a disadvantage in that the charge of a few ionic amino acids directly attached to C-terminus of protein of interest is masked by opposite charge of large protein of interest and so fusion protein cannot adsorb on the matrix efficiently. Thus the method has a low yield of purification.
In U.S. Pat. Nos. 5,179,196 and 5,322,930, the fusion peptides consisting of a fusion partner which is different from the protein of interest in the electric property, the protein of interest and CNBR cleavage site and Staph V8 cleavage site, respectively. However, the patents have a problem in that many proteins having a similar isoelectric point as well as the fusion protein can also adsorb on ion exchange chromatograph, and the cleavage method has a poor selectivity. Thus, the other proteins adsorbed on chromatograph are cleaved and solubilized together with the protein of interest.